Hepatitis E virus (HEV) was first discovered in 1983 as a cause of enterically transmitted hepatitis (Balayan et al., 1983). The full length viral genome was first cloned and sequenced in 1991, and it was found to be a single-stranded positive sense unenveloped RNA (Tam et al., 1991). Although morphologically resembling members of the Caliciviridae (Bradley et al., 1988; Huang et al., 1992; Panda et al., 1989), it has a distinct genomic organization (Berke et al., 1997). Based on sequence analysis, the 7.2 kb viral genome is predicted to contain three open reading frames (ORF) (FIG. 1) (Tam et al., 1991; Aye et al., 1992; Aye et al., 1993; Huang et al., 1992; Reyes et al., 1993). Non-structural viral proteins are encoded as a polyprotein by ORF1 located at the 5′ terminus of the viral genome. ORF2 is located at the 3′ end of the genome and encodes a major structural protein. The 5′ end of ORF3 has one base overlapping with the 3′ end of ORF1 and the 3′ end has 339 bases overlapping with the 5′ end of ORF2. ORF3 is believed to code for another structural protein whose function is still unknown. Linear antigenic epitopes have been located in ORF2 and ORF3 by epitope mapping and study of recombinant peptides (Coursaget et al., 1993; Khudyakov et al., 1993; Khudyakov et al., 1994).
Recent studies have shown that truncated recombinant peptides derived from both the amino and carboxy-termini of the major HEV structural protein of the ORF2 region can self-assemble into virus-like particles (Li et al., 1997). Although smaller in size, these virus-like particles were morphologically and antigenically similar to the capsid protein of the infectious virus (Li et al., 1997; Xing et al., 1999). Based on these findings, it would appear that the assembly of the recombinant peptide into the virus-like particles may have similar requirements as the assembly of the full length HEV structural protein into the larger capsid of the infectious virus particles. Notably, these findings suggest that the ORF2 encoded structural protein alone may be sufficient to self-assemble into the virus capsid. Moreover, it would appear that the interactions involved in the formation of the capsid protein per se may occur within a domain of the full length ORF2 protein from amino acid residues 112 to 608 (Xing et al., 1999). With a high isoelectric point of 12.35, the 111 amino acid long sequence located at the amino terminal domain of the major structural protein is believed to be involved in packaging of the viral genome (Briton and Heinz, 1990), however this domain evidently does not directly participate in the assembly of the virus capsid per se. The carboxy-terminal domain of the major structural protein also does not directly participate in the assembly of the virus capsid, however Li et al. (1997) have shown that appropriate proteolytic cleavage of this domain was essential for the assembly of virus-like particles.
Hepatitis E principally occurs in developing countries in both epidemic and sporadic forms. Several large outbreaks of hepatitis E occurred in the 1950's to 1980's caused by sewage-polluted drinking water (Visvanathan, 1957; Wong et al., 1980; Myint et al., 1985; Belabbes et al., 1985; Hau et al., 1999). The infection is usually self-limiting, but there are reports of serious complications when infection occurs during pregnancy (Tsega et al., 1992; Dilawari et al., 1994; Hussaini et al., 1997). One important preventive strategy employed in the combat against viral infection is the monitoring and detection of HEV in environmental specimens to ensure public health safety and environmental protection. However, traditional methods presently utilized for collecting and concentrating virus particles have several well known disadvantages that limit the investigation and discovery of a potential source of HEV. Two of the most common methods presently utilized are adsorption and centrifugation.
In the adsorption method, viruses are first concentrated from a test sample by adsorption to microporous filters and then eluted with large volumes of eluent. However, this technique also effectively concentrates a variety of other solutes, such as humic acids and proteins, which may interfere with the detection of viruses. In particular, many naturally occurring inorganic and organic solutes inhibit the nucleic acid polymerases useful in the amplification of target genomes (e.g. reverse transcriptase and Taq polymerase) (Tsai et al., 1992; 1993). The likelihood of nucleases and proteases being present in the test sample may degrade virus genomes before they can be amplified and therefore, contribute to an ambiguous result. In addition, various proteins, carbohydrates, and other organic compounds may bind magnesium ions and nucleotides necessary for the proper function of nucleic acid polymerases, while other solutes may exhibit toxic effects that could ultimately damage these polymerases (Demeke and Adams, 1992; Imai et al., 1992; Kolk et al., 1992).
In the centrifugation method, the test sample is homogenized and then centrifuged repeatedly. During the process, polyethylene glycol (PEG) is added to the supernatant and then centrifuged again. The final pellet is resuspended in buffer, however the final concentrate still contains toxic substances which may interfere with subsequent stages of the diagnostic method such as cell culturing, reverse transcription and polymerase chain reaction (Beril et al., 1996). As a consequence, it is necessary that the concentrate derived from the test sample undergo detoxification by gel filtration on a sephadex column.
Another important strategy useful in the fight against viral infection is the utilization of a vaccine for immunization against HEV to ensure public health safety and protection. However, it has not hitherto been possible to develop a vaccine against HEV infection using live attenuated or killed viral particles because of the difficulty in propagating the virus in cultured cells. As an alternative solution, it has been shown that some recombinant HEV peptides, especially those derived from the structural genes of the viral genome, are more promising in providing protection against contracting HEV (Tsarev et al., 1994a; Tsarev et al., 1997). Advantages to using recombinant peptides in vaccine formulations, compared to attenuated viruses, are that peptides can be more efficiently produce and conveniently purified. Furthermore, there is no possibility that the resulting vaccine will contain any live intact virus particles, thereby avoiding a potential risk of infectivity.
In view of the above comments, there remains a need for a reliable method of determining and diagnosing HEV which will not only avoid the disadvantages previously described, but also provide a more sensitive analytical test for detecting HEV in biological and environmental specimens. According to the present invention, there is provided a diagnostic method employing a highly immunoreactive recombinant viral peptide, pE2, derived from the carboxy-terminal end region of ORF2 of the hepatitis E virus (HEV) genome, which is proven to be highly reactive with sera from patients having current or past infection with HEV. Moreover, the pE2 peptide is antigenically related to the hepatitis E virus since part of its amino acid sequence is highly conserved among other HEV isolates. As described and further demonstrated herein, the antigenic properties of the pE2 peptide makes it an excellent candidate as a vaccine for the immunization and prevention of HEV infection. Accordingly, diagnostic methods useful in detecting HEV infection and a vaccine composition effective in preventing hepatitis E virus infection in which the novel pE2 peptide is utilized are provided herein.